Enhancing Coupled Enzymatic Activity by Colocalization on Nanoparticle Surfaces: Kinetic Evidence for Directed Channeling of Intermediates

Abstract

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by >50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to >100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.

Recent Advances in Enzyme-Nanostructure Biocatalysts with Enhanced Activity

“…There are many strategies for constructing multienzyme catalysis, such as co-immobilization of enzymes, conjugation of natural enzymes with artificial enzymes (i.e. nanozymes), and enhancing the function of enzymes within cell-free metabolic pathways [59,65,77,[93][94][95][96][97]. Encapsulation is a common form to maintain a high local concentration of enzymes and protect them from biological damage through proteases.…”

Quantum Dots and Gold Nanoparticles as Scaffolds for Enzymatic Enhancement: Recent Advances and the Influence of Nanoparticle Size

“…Enzymes can be immobilized either as single copies or as multiple enzymes which are part of a multienzyme cascade [4,[17][18][19][20][21][22]. Benefits of immobilizing single enzymes can include: (1) increased stability, (2) increased activity, (3) closeness and orientation to substrate, and (4) increased recoverability and reuse; whereas, the benefits of immobilizing multiple enzymes can include these plus: (5) increased (temporary) reaction rates, (6) bypassed intermediate toxicity, (7) bypassed offtarget pathways/directed catalysis, (8) reaction order, and (9) modularity ( Figure 1, Table 1) [4,10,[23][24][25][26][27][28][29][30].…”

“…In terms of benefits of immobilizing both single and multiple enzymes, enzyme immobilization can improve enzyme stability by keeping subunits in close proximity or affecting the local environment around the enzyme [17][18][19][20][21]. For example, the stability of an active tetrameric enzyme expressed as tagged, inactive monomers (tagged = enzyme with binding conjugation moiety epitope, e.g., His6-tag) will depend upon the equilibration between the associated and dissociated states; conjugating those tags to a scaffold can enhance association by keeping the monomers in close proximity [19]. Conversely, immobilization can decrease the stability of some enzymes through Reaction order Dictating order of reaction by choosing which enzymes to place in proximity 9 Modularity Modular tags allow interchangeability of enzymes to change cascade (and product) or make de novo cascades…”

“…Modeling, or empirical trials, can help determine the best location for conjugation tags and distance between enzymes and the scaffold [32][33][34][35][36][37]. Immobilization can also improve the catalytic activity of enzymes [4,19,[38][39][40][41][42][43][44]. For example, charged scaffold surfaces can modulate the local pH environment near an enzyme closer to its optimum, without the need to change buffer pH that may affect other processes [21,24,[45][46][47][48].…”

“…Overall, enzyme immobilization can offer inherent benefits, and these can be particularly exploited when working at the nanoscale with NPs. Advantages of NPs include: (1) multiple mechanisms for enzyme attachment; (2) high surface area to volume ratios and corresponding high radii of curvature leading to relatively large distances between enzymes and limiting detrimental protein-protein interactions; (3) the potential for multi-point attachment of (multi-subunit) enzymes, allowing in some cases increased enzyme stability; (4) the ability to structure solvent up to~2 × NP diameter, effecting many properties (e.g., pH gradients and boundary layers) that could affect substrate/intermediate/product partitioning; and (5) the ability to diffuse, whereas planar surfaces may have boundary layer and stagnation effects [19,22,42,[59][60][61][62][63][64]. While many NPs have been used for enzyme immobilization (silica, magnetic, silver, etc.…”